Ankyrins are large, widely distributed, tripartite proteins that link integral membrane transporters, adhesive proteins and receptors to the spectrin-based membrane skeleton. Three ankyrins have been cloned and characterized so far: Ank1 (erythrocyte ankyrin), Ank2 (brain ankyrin), and Ank3 (epithelial ankyrin). Each ankyrin contains an N-terminal "repeat" domain that attaches ankyrin to integral membrane proteins, a central spectrin-binding domain, and a C-terminal, "regulatory" domain, whose function is not characterized. Ank3 also exists in a smaller isoform (Ank3-5kb)that lacks the repeat domain. In macrophages Ank3-5kb is associated exclusively with late lysosomes and post lysosomal vesicles. The present studies aim to test aspects of ankyrin function in vivo, particularly whether Ank1 can be replaced by the other ankyrins and what the consequences are of replacing native Ank1 with an Ank1 bearing structural changes in the regulatory domain. These questions will be pursued using the technique of targeted gene replacement in mouse embryonal stem (ES) cells. The effects of the induced mutations on survival, organ function and pathology, red cell life span and morphology, red cell membrane and membrane skeletal composition and organization, membrane stability, and the function and mobility of band 3 will be investigated. Additional experiments will be directed at identifying sequences that target Ank3-5 kb to intracellular vesicles and identifying and cloning the target ligand(s).